Eukaryotic Kir Channels Research Articles

Structural and Functional Characterization of a Human Potassium Channel, Kir2.1

Inwardly rectifying potassium (Kir) channels are ubiquitously present in prokaryotic and eukaryotic cells. Kir channels selectively control the permeation of K+ ions in and out of the cell along the electrochemical gradient. Their inward rectification allows them to control the resting membrane potential and regulate many vital physiological functions such as heart rate, vascular tone, cell volume, and salt/fluid balance. Dysfunctions in Kir channels often lead to a multitude of diseases called “channelopathies”. Understanding the mechanisms involved in Kir channels and the effects of their mutations constitute a major challenge in the pharmaceutical and therapeutic research of the different channelopathies. In our case, we are interested in Andersen's syndrome, in which mutations of the human Kir2.1 protein are directly involved. By a biochemical, structural, and functional study, we aim to identify the differences between the wild type (WT) and two mutant channels in order to find links between the structure and the function of Kir2.1. This project aims at understanding the impact of clinically relevant disease-causing mutations on the structure and function of Kir2.1 potassium channels and to resolve the structure at high resolution using cryo-electron microscopy. The gating of Kir channels is modulated by various intracellular ligands that bind directly to the channel and enable its electrical activity. The lipid phosphatidylinositol-4,5-bisphosphate (PIP2) is essential to activate the eukaryotic Kir channels. We study a protein containing an arginine to histidine mutation in the putative PIP2 binding site and hypothesized that the loss of function observed in the mutated protein was due to a loss of interaction with PIP2. To test this hypothesis, we characterized the lipid-protein interaction using Surface Plasmon Resonance (SPR) and compared it with the WT protein. The SPR data shows that the mutant does not bind as well to the lipids as the WT.

Conformational changes at cytoplasmic intersubunit interactions control Kir channel gating

The defining structural feature of inward-rectifier potassium (Kir) channels is the unique Kir cytoplasmic domain. Recently we showed that salt bridges located at the cytoplasmic domain subunit interfaces (CD-Is) of eukaryotic Kir channels control channel gating via stability of a novel inactivated closed state. The cytoplasmic domains of prokaryotic and eukaryotic Kir channels show similar conformational rearrangements to the common gating ligand, phosphatidylinositol bisphosphate (PIP2), although these exhibit opposite coupling to opening and closing transitions. In Kir2.1, mutation of one of these CD-I salt bridge residues (R204A) reduces apparent PIP2 sensitivity of channel activity, and here we show that Ala or Cys substitutions of the functionally equivalent residue (Arg-165) in the prokaryotic Kir channel KirBac1.1 also significantly decrease sensitivity of the channel to PIP2 (by 5-30-fold). To further understand the structural basis of CD-I control of Kir channel gating, we examined the effect of the R165A mutation on PIP2-induced changes in channel function and conformation. Single-channel analyses indicated that the R165A mutation disrupts the characteristic long interburst closed state of reconstituted KirBac1.1 in giant liposomes, resulting in a higher open probability due to more frequent opening bursts. Intramolecular FRET measurements indicate that, relative to wild-type channels, the R165A mutation results in splaying of the cytoplasmic domains away from the central axis and that PIP2 essentially induces opposite motions of the major β-sheet in this channel mutant. We conclude that the removal of stabilizing CD-I salt bridges results in a collapsed state of the Kir domain.

Structural dynamics of potassium-channel gating revealed by single-molecule FRET.

Crystallography has provided invaluable insights to ion channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites, and examined structural dynamics within lipid membranes by single molecule FRET. We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and for cation channels in general.

Identification of a Cholesterol-Binding Pocket in Inward Rectifier K+ (Kir) Channels

Cholesterol is the major sterol component of all mammalian plasma membranes. Recent studies have shown that cholesterol inhibits both bacterial (KirBac1.1 and KirBac3.1) and eukaryotic (Kir2.1) inward rectifier K+ (Kir) channels. Lipid-sterol interactions are not enantioselective, and the enantiomer of cholesterol (ent-cholesterol) does not inhibit Kir channel activity, suggesting that inhibition results from direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionary conserved cholesterol-binding pocket, which we aimed to identify. Computational experiments were performed by docking cholesterol to the atomic structures of Kir2.2 (PDB: 3SPI) and KirBac1.1 (PDB: 2WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. The stability of cholesterol in each of these poses was then confirmed by molecular dynamics simulations. Finally, mutation of key residues (S95H and I171L) in this putative binding pocket found within the transmembrane domain of Kir2.1 channels were shown to lead to a loss of inhibition by cholesterol. Together, these data provide support for this location as a biologically relevant pocket.

Control of KirBac3.1 Potassium Channel Gating at the Interface between Cytoplasmic Domains

KirBac channels are prokaryotic homologs of mammalian inwardly rectifying potassium (Kir) channels, and recent structures of KirBac3.1 have provided important insights into the structural basis of gating in Kir channels. In this study, we demonstrate that KirBac3.1 channel activity is strongly pH-dependent, and we used x-ray crystallography to determine the structural changes that arise from an activatory mutation (S205L) located in the cytoplasmic domain (CTD). This mutation stabilizes a novel energetically favorable open conformation in which changes at the intersubunit interface in the CTD also alter the electrostatic potential of the inner cytoplasmic cavity. These results provide a structural explanation for the activatory effect of this mutation and provide a greater insight into the role of the CTD in Kir channel gating.

Phosphoinositide regulation of inward rectifier potassium (Kir) channels

Inward rectifier potassium (Kir) channels are integral membrane proteins charged with a key role in establishing the resting membrane potential of excitable cells through selective control of the permeation of K+ ions across cell membranes. In conjunction with secondary anionic phospholipids, members of this family are directly regulated by phosphoinositides (PIPs) in the absence of other proteins or downstream signaling pathways. Different Kir isoforms display distinct specificities for the activating PIPs but all eukaryotic Kir channels are activated by PI(4,5)P2. On the other hand, the bacterial KirBac1.1 channel is inhibited by PIPs. Recent crystal structures of eukaryotic Kir channels in apo and lipid bound forms reveal one specific binding site per subunit, formed at the interface of N- and C-terminal domains, just beyond the transmembrane segments and clearly involving some of the key residues previously identified as controlling PI(4,5)P2 sensitivity. Computational, biochemical, and biophysical approaches have attempted to address the energetic determinants of PIP binding and selectivity among Kir channel isoforms, as well as the conformational changes that trigger channel gating. Here we review our current understanding of the molecular determinants of PIP regulation of Kir channel activity, including in context with other lipid modulators, and provide further discussion on the key questions that remain to be answered.

Gating Motions of KirBac1.1 Cytoplasmic Domain with Respect to Transmembrane Domain Revealed by FRET

KirBac1.1 is a bacterial inward rectifier potassium (Kir) channel, which, contrary to its eukaryotic homologues, is strongly inhibited by phosphatidylinositol-4,5-bisphosphate (PIP2). The most recent crystal structures of eukaryotic Kir2.2 in complex with PIP2 indicate that the TM-CD linker forms a short a-helix in the presence of PIP2. As a result, the cytoplasmic domain of Kir2.2 moves about 6 angstrom towards the membrane surface. However, the ‘KKR’ motif in the TM-CD linker of Kir2.2, which directly interacts with PIP2, is absent in KirBac1.1 and the question arises: how does KirBac1.1 cytoplasmic domain move in response to PIP2 inhibition? In the present work, we have made KirBac1.1 tandem tetramer constructs and purified KirBac1.1 tandem proteins successfully. Reconstituted liposome flux assays indicate that the KirBac1.1 tandem protein remains functional, and retains sensitivity to PIP2 inhibition. We introduced two cysteine into the KirBac1.1 tandem tetramer, one at the extracellular loop of subunit 1 (G85 or T120) and one in the cytoplasmic domain of subunit 2 (A273). We labeled these cysteines with a FRET dye pair (Alexa-Fluor 488 and DABCYL) and measured FRET efficiencies in protein samples reconstituted into liposomes, in the absence and presence of PIP2. Our results indicate that the KirBac1.1 cytoplasmic domain moves ∼2-3 angstrom away from the transmembrane domain in the presence of PIP2 - opposite the direction suggested from eukaryotic Kir2.2 crystal structures in the presence and absence of PIP2. Reversed PIP2-dependent motions of the cytoplasmic domain with respect to the transmembrane domain between prokaryotic and eukaryotic Kir channels may explain their differential response to PIP2 modulation.

Identification of the Regulatory Anionic Lipid Site in Kir Channels

Inwardly rectifying potassium channels (Kir) are regulated by multiple factors, including multiple lipids. In addition to a specific requirement for phosphatidyl-4,5-bisphosphate (PI(4,5)P2) for channel activity of Kir2.1, analysis of purified proteins reconstituted into liposomes has revealed a secondary requirement for non-specific anionic lipids, which increase PI(4,5)P2 sensitivity by ∼100 fold [Cheng et al. 2011, Biophys. J. 100, 620-628]. Recent crystal structures of eukaryotic Kir channels in complex with PI(4,5)P2 reveal a common PI(4,5)P2 binding site [Hansen et al, 2011, Nature. 477, 495-498; Whorton & MacKinnon, 2011, Cell. 147, 199-208], but they have not identified the synergistic anionic lipid site.We have performed extensive docking simulations to identify potential interaction sites of different phospholipids with Kir2.1 channels. These simulations indicate two distinct binding sites; a high affinity site that corresponds to the crystallographic PI(4,5)P2 binding pocket and a lower affinity site, involving two lysine residues further towards the periphery of the cytoplasmic domain, that may correspond to the secondary anionic lipid site. When the two lysine residues are mutated to cysteine, channel activity is essentially abolished, even in the presence of PIP2. Cysteine modification of these residues by decyl-MTS, which essentially provides a ‘lipid tether' to the residue, restores channel activity in the presence of low levels of PIP2. These results point strongly to the identified site as being the site for non-specific anionic lipid interaction, and support a model in which the anionic lipid interaction (or ‘lipid tethering') pulls the Kir domain towards the membrane, facilitating PI(4,5)P2-mediated channel opening.

Identification of a Cholesterol Binding Pocket in Bacterial and Eukaryotic Inward Rectifier K+ (Kir) Channels

Cholesterol is the major sterol component of all mammalian plasma membranes. Recent studies have shown that cholesterol inhibits both bacterial (KirBac1.1 and KirBac3.1) and eukaryotic (Kir2.1) inward rectifier K+ (Kir) channels but the molecular basis of this interaction has yet to be determined. Biophysical experiments show that lipid-sterol interactions are not enantioselective, but only cholesterol and not the enantiomer of cholesterol (ent-cholesterol) inhibits Kir and KirBac channel activity, implying that the effects of cholesterol in both prokaryotic and eukaryotic channels must be due to direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer properties. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionarily conserved “cholesterol binding pocket”. To identify potential sites, computational experiments were performed in which cholesterol was docked to atomic structures of Kir2.2 (PDB: 3YJC) and KirBac1.1 (PDB: 3WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. Binding pockets that interacted with cholesterol and ent-cholesterol were eliminated. Residues identified as to solely interacting with cholesterol are being targeted for mutagenesis studies using purified channels. These experiments will identify a molecular basis for cholesterol-Kir channel interactions, and may provide a “model binding site” that can be used to identify putative cholesterol pockets in other channels.

Enantioselective Protein-Sterol Interactions Mediate Regulation of Both Prokaryotic and Eukaryotic Inward Rectifier K+ Channels by Cholesterol

Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Previous studies have shown that cholesterol inhibits inward rectifier K+ (Kir) channels, but have not distinguished whether this is due directly to protein-sterol interactions or indirectly to changes in the physical properties of the lipid bilayer. Using purified bacterial and eukaryotic Kir channels reconstituted into liposomes of controlled lipid composition, we demonstrate by 86Rb+ influx assays that bacterial Kir channels (KirBac1.1 and KirBac3.1) and human Kir2.1 are all inhibited by cholesterol, most likely by locking the channels into prolonged closed states, whereas the enantiomer, ent-cholesterol, does not inhibit these channels. These data indicate that cholesterol regulates Kir channels through direct protein-sterol interactions likely taking advantage of an evolutionarily conserved binding pocket.

The Lipid Dependence of Purified and Reconstituted Kir2.1 and Kir2.2

Ion channels are embedded in the membrane bilayer and are known to be regulated by their lipid environment. Insights on the structural basis of channel-lipid interactions have been gained by recent potassium channel crystal structures that reveal bound lipid or detergent molecules. However, efforts to define the lipid dependence of channel activity have been limited to cellular expression systems, in which the membrane composition cannot be fully controlled. We have expressed and purified functional human Kir2.1 and Kir2.2 from S. cerevisiae, and characterized the phospholipid dependence of channel activity in a liposomal 86Rb+ flux assay. Reconstituted Kir2.1 and Kir2.2 require incorporated PIP2 for activity and are maximally active in 0.1-1% PIP2 on a background of 3:1 POPE:POPG. This provides definitive evidence that eukaryotic Kir channels are directly activated by PIP2 without any intermediary components. Interestingly, Kir2.1 and Kir2.2 are minimally active in ∼1% PIP2 on a POPE (neutral) background, and are activated by increasing amounts of POPG (1 negative charge) or other anionic phospholipids. By contrast, the prokaryotic inward rectifier, KirBac1.1, shows no phospholipid dependence of activity, except potent inhibition by PIP2 (1), DGS-NTA, cardiolipin and oleoyl CoA. Our data suggest that the site of action for this secondary regulation by anionic phospholipids in Kir2.1 and Kir2.2 is distinct from the cytoplasmic PIP2 binding site. This study represents the first description of the lipid dependence of activity for recombinantly-expressed, purified eukaryotic ion channels in liposomes, and demonstrates that Kir2.1 and Kir2.2 have two lipid requirements for activity: a high affinity requirement that is specific for PIP2, and a low affinity requirement that is relatively non-specific for anionic phospholipids.1. D. Enkvetchakul, I. Jeliazkova, C. G. Nichols, J.Biol.Chem. 280, 35785 (2005).

The E71A Mutation Alters Selective Ion Permeability in KcsA

The mechanism of selectivity in potassium channels has been extensively studied using the prokaryotic potassium channel, KcsA. Computational studies suggest that a glutamate-aspartate H-bond behind the selectivity filter in KcsA may play a role in determining the permeation properties of the channel. However, the mutant E71A , which disrupts this H-bond interaction and abolishes pH-dependent inactivation is reported to have either no effect on K+ selectivity (1) or to increase K+ selectivity (2) as measured by reversal potentials. Using an 86Rb+ flux assay, WT KcsA exhibits strong K+ selectivity, such that there are no measurable 86Rb+ fluxes supported by Na+ and Li+. In contrast, both Na+ and Li+ support significant 86Rb+ fluxes in the E71A mutant, indicating an enhanced Na+ and Li+ permeability.In eukaryotic inward rectifying potassium channels (Kir), the E71 equivalent residue is part of a glutamate-arginine salt bridge that, when disrupted dramatically reduces K+ selectivity. KirBac1.1 is a prokaryotic channel that serves as a structural model of eukaryotic Kirs, but contains an H-bond in the equivalent position, similar to KcsA. By patch-clamping giant liposomes, we show that KirBac1.1 is K+-selective (PNa/PK < 0.08) as measured by reversal potentials shifts, but, like KcsA E71A, shows significant Na+ and Li+-driven 86Rb+ fluxes. We also find that the KcsA E71A mutant, similar to WT KirBac1.1. This loss of stability in these channels may suggest that the differences observed in permeation result from a weakened interaction with ions at the selectivity filter. Studies to examine ion permeation in eukaryotic Kir channels by 86Rb+ flux are ongoing.1. H. Choi, L. Heginbotham, Biophys.J. 86, 2137 (2004).2. J. F. Cordero-Morales et al., Nature Structural & Molecular Biology 13, 311 (2006).

KirBac1.1: it's an inward rectifying potassium channel.

KirBac1.1 is a prokaryotic homologue of eukaryotic inward rectifier potassium (Kir) channels. The crystal structure of KirBac1.1 and related KirBac3.1 have now been used extensively to generate in silico models of eukaryotic Kir channels, but functional analysis has been limited to 86Rb+ flux experiments and bacteria or yeast complementation screens, and no voltage clamp analysis has been available. We have expressed pure full-length His-tagged KirBac1.1 protein in Escherichia coli and obtained voltage clamp recordings of recombinant channel activity in excised membrane patches from giant liposomes. Macroscopic currents of wild-type KirBac1.1 are K+ selective and spermine insensitive, but blocked by Ba2+, similar to “weakly rectifying” eukaryotic Kir1.1 and Kir6.2 channels. The introduction of a negative charge at a pore-lining residue, I138D, generates high spermine sensitivity, similar to that resulting from the introduction of a negative charge at the equivalent position in Kir1.1 or Kir6.2. KirBac1.1 currents are also inhibited by PIP2, consistent with 86Rb+ flux experiments, and reversibly inhibited by short-chain di-c8-PIP2. At the single-channel level, KirBac1.1 channels show numerous conductance states with two predominant conductances (15 pS and 32 pS at −100 mV) and marked variability in gating kinetics, similar to the behavior of KcsA in recombinant liposomes. The successful patch clamping of KirBac1.1 confirms that this prokaryotic channel behaves as a bona fide Kir channel and opens the way for combined biochemical, structural, and electrophysiological analysis of a tractable model Kir channel, as has been successfully achieved for the archetypal K+ channel KcsA.

KirBac1.1: It's An Inward Rectifying Potassium Channel

The structures of KirBac1.1 and KirBac3.1 have been used extensively to generate in silico homology models of eukaryotic Kir channels and to explore ion permeation, gating and drug-channel interactions by computational approaches. Functional studies of KirBac1.1 have been limited to 86Rb+ flux assays, but we have now succeeded in measuring voltage-clamp currents of KirBac1.1 reconstituted in giant liposomes. Using the patch-clamp technique, we recapitulate the results of 86Rb+ flux experiments, showing that KirBac1.1 currents are potassium-selective, blocked by barium, and inhibited by PIP2. These findings suggest that KirBac1.1 channels are functionally similar to eukaryotic Kir channels. Like the weak inward rectifiers Kir1.1 and Kir6.2, introduction of a negative charge at the “rectification controller” residue in the inner cavity of KirBac1.1 (I138D) confers strong inward rectification (i.e. steep voltage-dependent block by spermine). Steady state single channel currents of KirBac1.1 show multiple subconductance levels and gating modes in 150 mM symmetrical K+. However, similar to eukaryotic Kir channels, single channel amplitudes exhibit mild intrinsic inward rectification, with a maximum conductance at ∼56 pS (-100 mV), and open probability is higher at positive potentials. Multiple conductance states are still present in single channel currents of other permeant ions such as Rb+ and Tl+. However, similar to many K+ channels, including KcsA, Rb+ and Tl+ single channel currents show increased mean open time and decreased conductance. We find that KirBac1.1(T142C), equivalent to the Kir6.2 high P(o) mutant L164C, also has a high open probability and is effectively blocked by Cd+. These electrophysiological results confirm that KirBac1.1 is a bona fide inward rectifying K+ channel and a tractable model for study of the molecular basis of inward rectification, permeation and gating in eukaryotic Kir channels.

Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

Potassium channels are tetrameric proteins that mediate K(+)-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (approximately 40 kDa) on SDS-PAGE. Addition of Ba(2+) (K(1/2) approximately 50 microm) prior to loading results in an additional tetramer band (approximately 160 kDa). Mutation A109C, at a residue located near the expected Ba(2+)-binding site, decreased tetramer stabilization by Ba(2+) (K(1/2) approximately 300 microm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba(2+). Neither mutation affected Ba(2+) block of channel activity (using (86)Rb(+) flux assay). In contrast to Ba(2+), Mg(2+) had no effect on tetramer stability (even though Mg(2+) was a potent blocker). Many studies have shown Cd(2+) block of K(+) channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd(2+) on KirBac1.1 with cysteine substitutions in M2. Cd(2+) block potency followed an alpha-helical pattern consistent with the crystal structure. Significantly, Cd(2+) strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba(2+), consistent with both ions simultaneously occupying the channel: Ba(2+) at the selectivity filter entrance and Cd(2+) coordinated by I138C side chains in the inner cavity.

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1.

ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.

Direct Modulation of Kir Channel Gating by Membrane Phosphatidylinositol 4,5-Bisphosphate

Multiple ion channels have now been shown to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) at the cytoplasmic face of the membrane. However, direct evidence for a specific interaction between phosphoinositides and ion channels is critically lacking. We reconstituted pure KirBac1.1 and KcsA protein into liposomes of defined composition (3:1 phosphatidylethanolamine:phosphatidylglycerol) and examined channel activity using a 86Rb+ uptake assay. We demonstrate direct modulation by PIP2 of KirBac1.1 but not KcsA activity. In marked contrast to activation of eukaryotic Kir channels by PIP2, KirBac1.1 is inhibited by PIP2 incorporated in the membrane (K(1/2) = 0.3 mol %). The dependence of inhibition on the number of phosphate groups and requirement for a lipid tail matches that for activation of eukaryotic Kir channels, suggesting a fundamentally similar interaction mechanism. The data exclude the possibility of indirect modulation via cytoskeletal or other intermediary elements and establish a direct interaction of the channel with PIP2 in the membrane.

Short variable sequence acquired in evolution enables selective inhibition of various inward-rectifier K+ channels.

Tertiapin (TPN), a small protein toxin originally isolated from honey bee venom, inhibits only certain eukaryotic inward-rectifier K(+) (Kir) channels with high affinity. We found that a short ( approximately 10 residues) sequence in Kir channels, located in the N-terminal part of the linker between the two transmembrane segments, is essential for high-affinity inhibition by TPN and that variability in the region underlies the great variation of TPN affinities among eukaryotic Kir channels. This short variable region is however not present in a bacterial Kir channel (KirBac1.1) or in many other types of prokaryotic and eukaryotic K(+) channels. Thus, the acquisition in evolution of the variable region in eukaryotic Kir channels has created the opportunity to selectively target the numerous types of Kir channel that play important physiological roles. We also show that TPN sensitivity can be readily conferred onto some Kir channels that currently have no known inhibitors by replacing their variable region with that from a TPN-sensitive channel. In heterologous expression systems, such acquired toxin sensitivity will allow currents carried by mutant channels to be readily isolated from interfering background currents. Finally we show that, in the heteromeric GIRK1/4 channels, the GIRK4 and not GIRK1 subunit confers the high affinity for TPN.